Trends in antimicrobial resistance of Shigella species in Peru, 2011–2020

Abstract Objective To describe the frequency of antimicrobial resistance rates and spatial-temporal distribution of Shigella species from the last 10 years in Peru. Methods A cross-sectional descriptive study was carried out. A total of 1668 Shigella strains, remitted as part of the national enteric pathogen surveillance from 2011 to 2020, were analysed. The strains were confirmed by conventional tests and serotyped with polyvalent and monovalent antibodies. Also, antimicrobial susceptibility was performed according to the Kirby–Bauer method. Results The most frequent Shigella species was S. sonnei (49.2%), followed by S. flexneri (42.2%), S. boydii (7.9%) and S. dysenteriae (0.7%). Phase II (46.29%) was the most frequent serotype in S. sonnei, serotype 2a (43.61%) in S. flexneri, serotype 2 in S. boydii and serotype 4 in S. dysenteriae. High rates of resistance were detected for trimethoprim/sulfamethoxazole (91.0%), tetracycline (88.4%), ampicillin (73.9%) and chloramphenicol (64.9%), moderate rates for amoxicillin/clavulanic acid (25.1%), ciprofloxacin (16.7%) and nalidixic acid (14.8%), and low rates for cefotaxime (1.74%), nitrofurantoin (0.7%) and ceftazidime (0.6%). Moreover, antimicrobial resistance to fluoroquinolones increased considerably from 2017 to 2020. Conclusion S. sonnei was the most frequent species, which have a large proportion of strains resistant to trimethoprim/sulfamethoxazole, and a growing trend of resistance to ciprofloxacin and nalidixic acid. This increase in resistance to commonly used antibiotics in treatments is alarming, threatening the control and management of these currently treatable infections.


Background
2][3] Shigellosis represents the second leading cause of death from diarrhoea in the world (212 400 deaths per year), 1,4,5 and one of the three leading causes of death from diarrhoea in children under 5 years of age. 4,6Shigellosis is an acute intestinal infection caused by the genus Shigella.It is divided into four serogroups with 47 serotypes: A (S. dysenteriae, 12 serotypes); B (S. flexneri, 15 serotypes), C (S. boydii, 18 serotypes) and D (S. sonnei, two antigenic types, phase I and phase II), 7,8 whose transmission is by the faecal-oral route, with an infective dose of 10-100 bacteria.Countries with a low socioeconomic level are mainly affected by this disease. 5,9,10ntibiotic therapy is indicated in patients with moderate or severe symptoms to reduce the duration and severity of the disease, the transmission and shedding of organisms and to prevent lethal complications. 5,9,100][11] Faced with this problem, the WHO recommended the use of ciprofloxacin as the first-line antibiotic for the treatment of all cases with bloody diarrhoea, and ceftriaxone and azithromycin as second-line antibiotics. 10urrently, Shigella strains resistance to ciprofloxacin has been increasing. 5,11Resistance to alternative drugs, such as ceftriaxone and azithromycin, has also been reported. 12,13If this trend continues, the management and control of shigellosis can be seriously affected.In addition to this problem, the scarcity of resources, the high rate of infectious diseases and the free access to antimicrobials would further complicate the management of shigellosis, reducing treatment options. 5,12,14In that sense, the

Study design and population
This is a retrospective and cross-sectional study.Shigella is routinely investigated in public and private health laboratories in Peru, but the notification in Peru is not mandatory.The strains are shipped to the Instituto Nacional de Salud when the other laboratories need confirmation of species, serotype and/or antimicrobial susceptibility.Therefore, the present study constitutes a passive report.
The population consisted of all Shigella strains remitted to the Instituto Nacional de Salud of Peru from 14 of the 25 regions of Peru, and recovered from 2011 to 2020.These strains were received by the National Reference Laboratory of Clinical Bacteriology under the framework of National Enteric Pathogens Surveillance.
To evaluate the distribution of Shigella strains over the time covered by the study, the date of collection of each strain were loaded into the R software v.4.3.1 to generate a violin plot using the ggplot2 library.

Culture and typing of Shigella species and serotypes
All Shigella strains were previously cultured in trypticase soy broth (Oxoid, England) for 6 to 8 hours; Subsequently, each strain was plotted onto Salmonella-Shigella agar (Oxoid, England) and incubated at 37°C from 18 to 24 h.Presumptive identification was carried out using biochemical tests consisting of triple sugar iron agar, lysine iron agar, mobility indole ornithine agar and Simmons citrate agar (Oxoid, England).The species and serotype were determined using commercially available polyvalent and monovalent antisera (Denka Seiken Co., Ltd, Japan).

Statistical methods
Descriptive statistics were used to analyse associations between the gender of the patients and their age or the Shigella species isolated, as well as AMR rates between the most frequent Shigella species.The differentiations between groups were analysed by Pearson's chi-square and a 95% confidence interval using IBM SPSS Statistic 23.
Of the total Shigella strains included in this study (n = 1668), 46.46% were isolated from male patients, 45.02% from females and 8.52% did not record data.Shigella isolation decreased with increasing age.From all species, the highest rate of isolation was observed in children from 0 to 5 years (67.33%),following a similar tendency by species, especially in S. sonnei, S. flexneri and S. boydii.An additional file shows this data in more detail (Table S1, available as Supplementary data at JAC-AMR Online).Moreover, no association was found between the gender of the patients and the age or the Shigella species isolated (P > 0.05) (Table 1).

Frequency of Shigella species and serotypes
The predominated Shigella species in this study was S. sonnei with 49.2% (n = 821), followed by S. flexneri with 42.2% (n = 704), S. boydii with 7.9% (n = 131) and S. dysenteriae with 0.7% (n = 12).According to the temporal analysis, the recovery rate of S. sonnei was constant, with little variations from 2011 to 2020, while in the other three species, their recovery rate was heterogeneous in the same period of time (Figure 2).Also, no strain of S. dysenteriae was recorded in the years 2015, 2016, 2019 and 2020.In fact, S. dysenteriae was the species with the lowest frequency in this study.
Resistance rates in S. boydii to trimethoprim/sulfamethoxazole, tetracycline and ampicillin were 93.1%, 84.0% and 64.8%, respectively.Trimethoprim/sulfamethoxazole resistance rates in S. boydii and S. dysenteriae were higher than in S. flexneri.In addition, a statistically significant association was found between AMR to trimethoprim/sulfamethoxazole, tetracycline, chloramphenicol, ciprofloxacin and nalidixic acid in S. sonnei and S. flexneri (P < 0.05) (Table 3).
Moreover, notable differences in antibiotic resistance profiles were also observed, especially those that changed significantly from 2011 to 2020.In S. sonnei, the amoxicillin/clavulanic acid resistance rate was decreasing from 2014 to 2020, while the resistance of ampicillin and chloramphenicol decreased during the 2017-2020 period.However, resistance to ciprofloxacin and nalidixic acid increased considerably from 2017 to 2020.On the other Antimicrobial resistance of Shigella species in Peru, 2011-2020 hand, the rate of resistance to tetracycline, ampicillin and chloramphenicol in S. flexneri decreased moderately during 2017-2020, while ciprofloxacin resistance increased moderately (Figure 3).Specific details are shown in an additional file (Table S2).
Additionally, 96.5% of S. sonnei were MDR, presenting resistance to more than two classes of antibiotics, while 1.2% and 2.1% were resistant to two and one antibiotics, respectively.In S. flexneri, 74.4% were resistant to more than two antibiotics, while 22.6% 7.8% were resistant to two and one antibiotics, respectively.In S. boydii, 64.9% were resistant to more than three antibiotics, while 24.4% and 9.2% were resistant to two and one antibiotics.An additional file shows this in more detail (Table S3).

Discussion
Despite the efforts made to control bacterial diarrheal infections, Shigella is one of the leading causes of bacillary dysentery in Peru, Latin America, and other developing countries around the world. 1,4,5The age groups with the highest prevalence of shigellosis in these regions were children, especially those belonging to the age range from 0 to 5 years, 1,2 which was reflected in this investigation (Table S1).Other age groups such as adults can also be affected by this disease, but the frequency rate in countries such as Peru is usually low, compared to the observed in children 18,28 ; a situation different from that observed in developed countries such as the USA or England, which in recent years have reported an increase in shigellosis in adults; however, the way in which they acquire this infection is through sexual contact. 29,30ne of the main limitations of this study is that most of Shigella strains were isolated in Lima (90.29%) (Figure 1).This is because many of the regions of Peru have limitations to carry out the diagnosis of Shigella by stool culture, often using traditional laboratory tests such as the presence of faecal leukocytes. 31For this reason, it is possible that the AMR rates presented in this analysis do not contemplate the real dimension of AMR at the level of the entire country.However, the number of strains included to achieve this study, as well as the period of time that it covers, is higher than those carried out by previous authors in Peru. 18,28urrently, several epidemiological studies have reported a change in the proportion of Shigella species around the world.In a study conducted in six Asian countries, S. flexneri was the most frequently isolated species in Bangladesh, China, Pakistan, Indonesia and Vietnam, while S. sonnei was the most predominant in Thailand. 6The changing epidemiology of shigellosis in developing countries located in Asia, Latin America and the Middle East shows a proportional decline of S. flexneri, with the simultaneous appearance of S. sonnei. 179][20] However, some changes have been observed in recent years.Guevara et al. were the first to describe the increased frequency of S. sonnei. 21On the other hand, Riveros and Ochoa found that 50% of the Shigella strains recovered since 2008 in the US Navy Medical Research Unit No. 06 (NAMRU-6) were identified as S. sonnei. 22In this study, a constant and significant increase in the frequency of S. sonnei (49.2%) in relation to S. flexneri (42.2%) was observed in a period of 10 years (Figure 2); a similar trend that has also been observed in previous studies. 12,32,33The increase in the isolation of S. sonnei was reported mainly in countries and regions that experienced rapid industrialization and sanitation development, 17 as occurred in several Latin American countries where new economies emerged and growing industrialization contributed to improving water sanitation. 34In the last two decades, Colombia reports a  Quino et al.
constant increase of S. sonnei (52.7%) compared to S. flexneri (43.9%), 35 while Brazil shows a marked predominance of the frequency of S. sonnei (88.2%) against S. flexneri (11.8%). 36he observed contradiction between the increase of S. sonnei versus better sanitation has not been determined, however, several hypotheses have been proposed. 17A possible explanation says S. sonnei and Plesiomonas shigelloides share a common O antigen, which may lead to natural cross-protective immunity in populations exposed to both microbial populations. 37These influences could have led to important changes in infectious disease patterns and epidemiological conditions, which could explain the observed change in the prevalence of S. sonnei. 38n the other hand, there is a possibility that the observed increase in S. sonnei is due to a greater capacity for speciation and clinical reporting. 38In addition, a greater survival and replication of S. sonnei inside Acanthamoeba has been reported, being able to act as a reservoir, which would favour a greater capacity to acquire AMR. 39As more countries increase their level of development and sanitation, S. sonnei could become a serious global public health problem.
On the other hand, the prevalence of S. boydii (7.9%) and S. dysenteriae (0.7%) were low compared to S. sonnei and S. flexneri.These findings agree with the low frequency of S. boydii reported in our country previously, 11 however, it is frequently reported in Southeast Asia.Moreover, S. dysenteriae has not been reported in Peru for several years, except for the study conducted by Kosek et al., which in 2008 reported a frequency of 2.4% in the Peruvian Amazon. 19S. dysenteriae is most common in outbreak settings associated with civil unrest and refugee crises. 40,41egarding the Shigella serotypes identified in this study, phase II was predominant in S. sonnei, which is consistent with some previous studies. 42On the other hand, eight different serotypes were observed in S. flexneri (1, 2, 3, 4, 5, 6, variants X and Y), being serotype 2a the most frequent (43.61%); this data coincides with two studies carried out in Peru, one by Koseck et al. 19 and another by Guerrero et al., 20     Antimicrobial resistance of Shigella species in Peru, 2011-2020 corresponded to serotype 2a, respectively.Similar data were reported in Uruguay 43 and Colombia, 44 with 68% and 40% of cases, respectively.
Although Shigella serotypes share similar properties, they exhibit unique epidemiological characteristics.The true reason for the predominance of a serotype over a region is still not known with certainty. 45However, it is noted that some serotypes have a greater number of genetic virulence factors, such as S. dysenteriae serotype 1, which would influence their high mortality rate. 46dditionally, there is evidence that some of the serotypes are more likely to cause extraintestinal diseases, such as blood stream infections (S. dysenteriae serotype 1 and S. flexneri 3a), 47 reactive arthritis (S. flexneri serotype 1, 2 and 2a, S. sonnei and S. dysenteriae type 1) 48 and Hemolytic uremic syndrome (S. dysenteriae serotype 1). 49For all of these reasons, it is possible that the presence of specific S. flexneri or S. sonnei serotypes in our country increases the risk of causing an extraintestinal disease; however, this study only focuses on diarrheal manifestations.
The constant increase in AMR of Shigella species is a major problem in the treatment of Shigella gastroenteritis, especially the MDR strains. 50One of the main concerns surrounding Shigella is its ability to rapidly acquire antibiotic resistance, particularly S. sonnei, which can acquire resistance genes directly from E. coli through horizontal gene transfer.Several recent reports have suggested that S. sonnei is capable of sharing resistance plasmids through conjugation with commensal E. coli. 51,52In our study, approximately 84.2% of all Shigella strains showed MDR profiles, which is significantly higher than the rate of 41.6% (1762/4234) from the NARMS report (2005-2014). 53All MDR strains were highly resistant to traditional antimicrobials such as trimethoprim/sulfamethoxazole, tetracycline, ampicillin and chloramphenicol.This confirms that therapeutic alternatives for Shigella infections are scarcer, forcing the use of other groups or new antimicrobials.The reasons for the rapid accumulation of resistance are the excessive or inappropriate use of antibiotics in outpatients. 54,55higella resistance to trimethoprim/sulfamethoxazole, tetracycline, ampicillin and chloramphenicol has been reported by previous studies, particularly in S. flexneri and S. sonnei, 18 a trend that continues until recent years, according to our analyses (Table 3).Something interesting is the decrease of AMR in S. sonnei to ampicillin and chloramphenicol during the years 2019-2020, with the consequent increase in resistance to fluoroquinolones (Figure 3).Since the acquisition of Shigella AMR to each of these antibiotic groups is attributed to different mechanisms, it is necessary to perform sequencing studies to understand the causes of such variation. 50luoroquinolones and third-generation cephalosporins are the alternative and first-line drugs recommended by the World Health Organization for the empirical treatment of shigellosis. 56owever, in recent years, resistance to fluoroquinolones (ciprofloxacin and nalidixic acid) has increased significantly in all Shigella species, especially S. sonnei and S. flexneri. 13,38,50In this study, resistance to ciprofloxacin and nalidixic acid has increased considerably in S. sonnei since 2017, while the increase was moderate in S. flexneri and S. boydii.Regarding cephalosporins, resistance was less than 1.7% of the total strains analysed.However, our study shows that current resistance patterns have changed; several investigators suggest that empirical therapy should be modified according to these changes, and that treatment should be based on susceptibility patterns. 50urveillance of AMR to azithromycin as a second-line antibiotic used to treat cases of shigellosis in Peru has been actively carried out since 2019.The lack of data corresponding to the years 2011-2018 did not allow a detailed analysis with this antibiotic, constituting the second limitation of our study.However, the data collected during 2020 (Table S4) suggest a high rate of resistance, especially against S. sonnei (65.1%) and S. flexneri (43.9%), a proportion comparable to that observed in other countries. 13,57

Conclusions
In conclusion, our findings show the predominance of S. sonnei in relation to S. flexneri, as well as a large proportion of resistance to trimethoprim/sulfamethoxazole, an antibiotic considered the first drug of choice to treat patients with inflammatory diarrhoea caused by Shigella.Also, an increasing trend in resistance to ciprofloxacin and nalidixic acid was observed.This increase to fluoroquinolones resistance is alarming and threatens the ability to control and manage this currently treatable disease.Finally, the implementation of an active Shigella surveillance is necessary in Peru and other developing countries to reinforce the prevention and control measures of shigellosis, Also, it is necessary to incorporate the use of molecular tools including Next Generation Sequencing for identification of novel mechanisms of AMR and the emergency of MDR Shigella strains.

Figure 1 .
Figure 1.Map of Peru indicating the geographical distribution where Shigella strains were isolated.The size of the nodes indicates the number of species recovered in each region (according to the scale), while colors indicates Shigella species according to the legend.At least, one clinical strain from 14 Peruvian regions was recovered for this study.

Figure 2 .
Figure 2. Temporal distribution of Shigella species by year of isolation in Peru.S. flexneri and S. sonnei have been the most frequent species throughout the study period, followed by S. boydii.The recovery rate of S. sonnei was constant, in contrast to the other three species that were heterogeneous.

Table 1 .
Chi-square test results on the relationship of age and Shigella species with the gender of patients

Table 2 .
Distribution of Shigella serotypes by species from 2011 to 2020
a The total includes S. dysenteriae.b Comparison between S. sonnei and S. flexneri.c P < 0.05.Quino et al.